Methobottromycin, process for preparing and using same

ABSTRACT

This invention relates to a new antibiotic agent, methobottromycin, its salts, the process of preparing same, and their use as an antibiotic agent in the treatment of chronic respiratory disease in chickens and infectious sinusitis in turkeys.

Umted States Patent H 1 3,683,073 Wolf et al. [451 Aug. 8, 1972 [54]METHOBOTTROMYCIN, PROCESS FOR PREPARING AND USING SAME [56] ReferencesCited [72] Inventors: Frank J. Wolf, Westfield; William J. OTHER puguc'no s Mille S t; LOUB' 'g flfi sg Derwent Farmdoc No. 25,373,Abstracting we 66, l l5l3, published Feb. 17, I967. [73] Assignee: Merck& Co., Inc., Rahway, NJ.

22 F! d: M h 14 Primary Examiner-Jerome D. Goldberg 1 I e m 1968Attorney-Reverdy Johnson and 1. Louis Wolk [21] Appl. No.: 713,255

Related US. Application Data :7 I ABS CT b is invention re ates to a newanti iotic agent, [63] ggg g of methobottromycin, its salts, the processof preparing l a domed same, and their use as an antibiotic agent in thetreatment of chronic respiratory disease in chickens and in- [52] US. Cl..424/1l7, 195/80 51 Int. Cl. ..A6lk 21/00 fect'ous m turkeys Field ofSearch ..424/ 1 17; 195/80 11 Claim, 1 Drawing Figure METHOBATHOMYCIN,PROCESS FOR PREPARING AND USING SAME This application is a continuationof Ser. No. 480,039, filed Aug. 16, 1965, now abandoned.

This invention relates to a new antibiotic agent, its salts, the methodof preparing same and their use. More particularly, the presentinvention is concerned with a novel, highly active antibiotic compoundknown as methobottromycin, its salts, the process of preparing same, andtheir use as an antibiotic agent in the treatment of chronic respiratorydisease in chickens and infectious sinusitis in turkeys.

The discovery of remarkable antibiotic properties of penicillin andsimilar substances has stimulated great interest in the field ofantibiotic compounds such as: streptomycin, gramicidin, subtilin,bacitracin, chlortetracycline, oxytetracycline, cycloserine, colistinfervenulin, streptozotocin, novobiocin and the like. In general, suchantibiotics are particularly active against certain gram positivebacteria. Others are active against gram negative bacteria and some areactive against both gram negative and gram positive bacteria. However,the activity of these known antibiotics is usually limited to a fewpathogenic microorganisms and work has been conducted in this field inan attempt to find additional antibiotic substances which would beeffective against other pathogens.

In addition, many bacteria which, at one time, were controlled by knownantibiotics, have developed increasing resistance over the years tothese antibiotic substances. As a result, although some of theseantibiotics have been found to be invaluable in the treatment of variousdiseases, it has been discovered that certain strains of some pathogensdevelop a resistance to various particular antibiotics and,consequently, these antibiotics are no longer active against suchstrains of pathogens or the activity of these antibiotics has beenreduced to such a degree so as to make their use against such pathogensof little consequence.

Accordingly, the deficiencies of the known antibiotics have stimulatedfurther research to find other antibiotics which will be highly activeagainst a wider range of pathogens as well as those strains of variousmicroorganisms which are resistant to other antibiotics. This is truenot only with disease-producing bacteria which attack humans but alsofor disease-producing bacteria which attack animals and poultry.

Chronic respiratory disease is a disease of chickens and turkeys, causedby a certain group of microorganisms known as PPLO orpleuropneumonia-like organisms, which have been classified asMycoplasma. This is referred to in the art as PPLO infection. inchickens the disease may be complicated by a secondary invader, at whichtime the disease is known as chronic respiratory disease complex. Inturkeys this disease appears in two forms. It is called infectioussinusitis when it is in the form that affects the upper respiratorytract, and air sac disease when it affects the lower respiratory areas.For the purpose of simplicity, these diseases will be referred to hereinas infectious sinusitis.

In chickens the chronic respiratory disease symptoms may be like thoseof any other respiratory disease such as Newcastle disease, infectiousbronchitis, laryngotracheitis, fungus infection, etc. The usuallyobserved symptoms are nasal discharge and a slight swelling below theeye. Coughing, sneezing, and a hoarse throat rattle or rale mayaccompany these signs. The symptoms of the disease in turkeys is oftendemonstrated by swollen sinuses with gelatinous exudate, watery eyes andcoughing. The air sacs and respiratory passages will be plugged bycheesey exudates.

The economic loss that accompanies chronic respiratory disease is a dropin egg production by at least 10 to 40 percent, which afi'ects the birdsfor several weeks or months. Poor hatchability of fertile eggs laid byinfected hens can cause additional losses. Mycoplasma (PPLO) causedinfection results in the death of a high percentage of embryos. Loss ofweight in a large percentage of birds is also evident. There is, inaddition, a significant amount of mortality in birds beginning at aboutfour weeks of age.

Infection of birds may occur in a number of ways. Birds may be infectedby contact with other infected birds, usually by an inhalation of nasalexudate from a sneezing bird. In fact, infected chickens or turkeys maybecome sick, and they may become carriers in which they appear to behealthy but are, in fact, infected with pathogenic strains of Mycoplasma(PPLO). In addition, birds may be infected through contaminated litter,manure, water and feed, breeding hens or contaminated hatcheries.Transmission of the disease via an infected embryonated egg contributeslargely to an infected flock.

Chemotherapeutic control of these diseases has been successful with avery limited number of compounds. With one exception, the agents whichhave been found satisfactory are known antibiotics used clinically forother diseases, principally human diseases. The exception is theantibiotic tylosin. Although tylosin is used fairly broadly, strains ofPPLO resistant to it have been encountered, and the antibiotic has beenshown to be toxic in use with turkeys.

Other antibiotics useful for controlling chronic respiratory disease inchickens and infectious sinusitis turkeys are erythromycin andchlortetracycline or oxytetracycline. However, the dosage levels ofthese antibiotics required to obtain good results are quite high, whichresults in an economic barrier to the user. Other antibiotics known tohave anti-PPLO activity usually require a dosage level too close to thetoxic level to be of practical value. Included in this group areneomycin, kanamycin, and chlorarnphenicol.

' Many other antibacterial antibiotics which are used for otherinfections have been found to be without effect on the PPLO. Examples ofthese would include penicillin and its many derivatives, cycloserine,

novobiocin, and many others. As can be seen, this group includes agentswith a wide spectrum of activity, hence their inactivity againstMycoplasma shows how these microbes are a unique and specialized type ofbacterium.

It is an object of the present invention to provide useful antibioticsubstances which are highly effective in controlling the primaryetiologic agent of chronic respiratory disease of chickens andinfectious sinusitis of turkeys.

An additional object of the present invention is to produce a new anduseful antibiotic substance which may be used in higher concentrationsthan those presently available without the resultant danger of toxicity.e

Another object of the present invention is to provide an antibiotic thathas an acceptable oral absorption for treating chronic respiratorydisease of chickens and infectious sinusitis of turkeys.

A further object of the present invention is to provide an antibioticthat may be applied in relatively low dosages in the treatment ofchronic respiratory disease of chickens and infectious sinusitis ofturkeys.

Another additional object of the present invention is to provide anantibiotic that is active against a wide range of strains ofmycoplasmas, including those belonging to the species M. gallisepticum(PPLO), in the treatment of chronic respiratory disease of chickens andinfectious sinusitis of turkeys.

A still further object of the present invention is to provide a processof preparing this novel antibiotic substance.

Other additional objects of the present invention will become apparentto those skilled in the art by reading the following specification.

The new antibiotic substances of the present invention are found bygrowing, under controlled conditions, a previously unknown species ofmicroorganism. The

microorganism was isolated from the fermentation broth of a soilactinomycete collected from Canada.

This new microorganism has been designated Strepto- STREPTOMYCESCANADENSIS MA-959 Morphology Biverticillate. Straight chains of eight tospores, a few chains longer. Spores cylindrical (950 X Avg. size 1.0 Xl.7p..)

Czapek Dox Agar (Sucrose Nitrate) Growth light. Aerial mycelium scant,white. Vegatative growth colorless. Reverse colorless. No solublepigment. Sporulation good.

Glycerol Aspargine agar Growth good. Aerial mycelium medium gray.Vegatative growth brown to reddish-brown. Reverse brown to reddishbrown.Soluble pigment brown to reddish-brown (pink in early stage of growth 1week). No sporulation observed.

Tomato paste Oatmeal agar Growth good. Aerial mycelium medium gray withwhite tufts and pink exudate appearing after 3 weeks. Vegetative growthbrown. Reverse dark brown. Soluble pigment brown. Some sporulation.

Emersons agar Growth moderate. Aerial mycelium scant, light gray.Vegetative growth brown. Reverse brown. Soluble pigment light brown(pink in early stage).

Potato plug Growth good. Colonies smooth, cream to grayish-brown. Aerialmycelium light gray (appears only in drier portion of plug). Solublepigment medium brown (pink in early stage).

Starch agar Growth good. No aerial mycelium. Vegetative growth lightbrown. Reverse light brown. Soluble pigment light brown (pink in earlygrowth stage). Hydrolysis.

Nutrient Gelatin plate Growth good. No aerial mycelium. Vegetativegrowth light brown. Reverse light brown. Soluble pigment light brown.Liquefaction.

Gelatin stab Soluble pigment dark greenishbrown. One-third liquefaction.

Calcium Malate agar Growth good. Aerial mycelium scant, pinkish white.Vegetative growth yellowish brown. Slight browning of medium alonggrowth streak.

Tyrosine agar Growth moderate. Aerial mycelium pale pinkish-white.Vegetative growth very light brown. Slight browning of medium.

Peptone-lron Yeast Extract slant Growth good.

No aerial mycelium. Vegetative growth gray. Soluble pigment blue-blackat 2 days. Medium brown at 3 weeks.

Skim Milk agar Growth good. Aerial mycelium pinkish white. Vegetativegrowth light brown. Soluble pigment very light brown. No hydrolysis.

Reduction of nitrates Negative under test conditions in organic andsynthetic media.

Temperature Good growth at 28 C. No growth at Micro-aerophilic growth(yeast extract-dextrose stab) Heavy surface growth and along two-thirdsof stab line.

Milk Peptonization complete at 3 weeks. No coagulation. Heavy brownishgrowth ring with sparse aerial mycelium (light gray). Soluble pigmentmedium grayish-brown. Alkaline reaction (pH 7.9).

Litmus Milk Peptonization complete in 3 weeks.

No coagulation. Alkaline reaction.

The above description of the microorganism-producing methobottromycin isgiven as illustrative of suitable strains of Streptomyces which can beused in the production of methobottromycin, but it is understood thatthe information herein described is not to be limited to organismsanswering this particular description. The present invention alsocontemplates the use of other species of Streptomyces or mutants of thedescribed organisms such as those obtained by natural selection or thoseproduced by mutating agents, for example, X-ray irradiation, ultravioletirradiation, nitrogen mustards, and the like.

The new antibiotic of the present invention is a basic compound formingsalts with acids, both inorganic and organic, such as hydrochloric,tartaric, salicylic, etc., and other compounds. The free base form ofmethobottromycin possesses the following physical and chemicalproperties:

a. Crystallizes from ethyl acetate in the form of white prisms meltingat a temperature of from about 166 to 167 C.

b. Easily soluble in alcohols, esters, ethers,

chlorinated solvents and benzene.

c. Partly soluble in water.

(1. Insoluble in petroleum ether, hexane and the like.

e. Has a specific rotation of ]n 15 in a 5 percent solution of percentethanol.

Unfortunately, in contrast to other antibiotics,

methobottromycin is found to be an exceedingly complex compound having amolecular weight of about 800. Consequently, thus far it has not beenpossible to determine the exact complete composition ofmethobottromycin. It has been found that this composition contains theelements carbon, hydrogen, nitrogen, sulfur and oxygen. The foundpercentages of these elements are as follows:

C 59.50% H- 7.52% N 13.50% S 3.90%

15.58% (by difference) Total 100.00%

These data suggest a molecular structure C H N O,S, but other similarmolecular formulae are possible within the experimental error of thesedeterminations.

The infrared absorption spectrum of the antibiotic methobottromycin inchloroform using sodium chloride prism is illustrated in theaccompanying drawing. The more significant of the characteristic peaksoccur at the following wave lengths expressed in reciprocal centimeters:3,300, 2,950, 1,730, 1,6301,650, 1,490, 1,360, 1,300, 1,242, 1,160,1,132, 1,119, 1,102, 980, 806.

The above infrared spectrum readings can be more clearly seen in theattached drawing. Upon acid hydrolysis, methobottromycin yields sixninhydrin-positive substances, none of which include proline.

Methobottromycin exhibits characteristic R; values in the followingsolvent systems:

(In all cases paper was wetted with stationary phase.)

Characteristics of the antibiotic of the present invention may alsoinclude thin layer chromatography. Thin layer chromatographic platescontaining silica gel are developed in 94 percent chloroform and 6percent methanol, dried and placed in a chamber containing iodine vapor.A brown stain indicates the presence of the antibiotic of the presentinvention, methobottromycin. The R, of this zone is 0.64.

The culture producing methobottromycin produces generally two types ofsubstances: a netropsin-type antibiotic and a bottromycin-typeantibiotic. The bottromycin group of antibiotics from whichmethobottromycin is extracted, is readily separated from the netropsingroup by extraction with chloroform from aqueous solutions. Thechloroform extract, after purification, shows the presence of fiveantibiotic substances on bioautograph of paper strips. The paper stripsystem utilized for this test consists of paper impregnated with caprylalcohol and developed downflow with buffer, wherein the R, ofmethobottromycin is 0.19. The five components have been designatedcomponents A through E in order of decreasing polarity. It issignificant that methobottromycin demonstrated considerably greaterantibiotic activity than any of the other components. Table A belowlists bioactivity of all of the components of bottromycin. The firstcolumn Staph. MIC" is a tube dilution assay which measures the minimalinhibitory concentration of the antibiotic in a broth culture of thetest microorganism, Staphylococcus aureus. The second column In Ova EDis an in ova assay which measures the effective dose level of theantibiotic in protecting an e'mbryonated egg from a known infection ofmicroorganism, in this .case, Mycoplasma gallisepticum (PPLO). Thisassay is modeled after the test system described by R. Yamamoto and H.E. Adler (Am. J. Vet. Res., July .1965) except that the parameter usedto measure effectiveness of the drug was protection against mortalityinstead of prolongation of the embryos life.

TABLE A In Ova Component Staph. MIC pg/ml ED, #g/egg B 0.23 13(Amethobottromycin) C 0.04 5 (Methobottromycin) Table B shows thatninhydrin-producing substances are liberated from the five components onacid hydrolysis and paper chromatography.

percentage present too small to separate As can be seen from Table B,methobottromycin contains methyl proline but does not contain proline.

In an aqueous solution at pH greater than 10, the antibiotic isunstable. However, at pH from 3 to 9 the product is stable for 24 hoursat room temperature.

Methobottromycin is active in inhibiting gram positive microorganismsprimarily although it exhibits some activity against gram negativemicroorganisms.

The new antibiotic of the present invention is produced by the aerobicfermentation of Streptomyces canadensis MA959 in a suitable aqueousmedium. Aqueous mediums such as those employed for the production ofother antibiotics are suitable for the production of methobottromycin.Such mediums contain sources of carbon and nitrogen, assimilable by themicroorganism, and inorganic salts. In addition, the fermentationmediums contain traces of metal necessary for the growth of themicroorganism which are usually present in complex sources of carbon andnitrogen in the medium.

In general, carbohydrates such as sugars, for example, dextrose,sucrose, dextrin and the like, are suitable sources of assimilablecarbon. The exact quantity of the carbon source will depend, in part,upon the other ingredients of the medium, but it is usually found thatan amount of carbohydrate between about 1 and 6 percent by weight of themedium is satisfactory. These carbon sources can be used individually,or several such sources may be combined in the medium.

Various nitrogen sources such as casein hydrolysates, amino acids, forexample, asparagine, glycine, arginine, digests of soybean meal, soybeanmeal, distillers solubles, and the like are readily assimilated by themethobottromycin producing microorganisms and can be used infermentation mediums for the production of this antibiotic. In general,we find that organic sources of nitrogen, particularly soybean meal, arevery satisfactory for the production of the new antibiotic. The variousorganic and inorganic sources of nitrogen can be used either alone or incombination in amounts ranging from about 0.2 percent to about 6 percentby weight of the aqueous medium.

The following example illustrates a method of preparing the antibioticof the present invention, but it is to be understood that it is givenfor purposes of illustration and not of limitation.

EXAMPLE 1 A. Fermentation A medium containing 1 percent dextrose, 0.3percent meat extract, l.0 percent tryptic digest of casein, and 0.5percent sodium chloride was made up in water and adjusted to pH 7.0 withsodium hydroxide, sterilized and aseptically added to a slant culture ofStreptomyces canadensis MA-959 (ATCC 17776) and the spores scraped intosuspension. About 3 ml. of this spore suspension was aseptically addedto a stoppered 2 liter baffled Erlenmeyer flask containing 500 ml. ofsterile aqueous medium consisting of 1 percent dextrose, 0.3 percentmeat extract, 1.0 percent tryptic digest of casein, and 0.5 percentsodium chloride and the pH again adjusted to 7.0. The flask wasincubated at 28 C. on a rotary shaker at a speed of 120 RPM with a2-inch throw for a period of 48 hours.

This vegetative culture was then aseptically added to a 50-gallonstainless steel fermenter containing about 30 to 40 gallons of sterilemedium having a composition comprising 1.5 percent yeast autolysate, 1percent dextrose, 0.25 percent sodium chloride with the pH adjusted to7.5. The inoculated medium was incubated at 28 C. for 40 hours duringwhich time it was agitated with sterile air being passed through themedium at a rate of about 3 cubic feet per minute. About 8.4 percent ofthis vegetative culture was employed to inoculate a ISO-gallon stainlesssteel fermenter containing about gallons of a medium having thecomposition comprising 1.5 percent yeast autolysate, 0.5 percent sodiumchloride, and 3 percent dextrose at pH 7.0 previously sterilized withsteam at about l20 C. for 15 minutes. The culture was incubated at 28 C.with agitation and aeration at a rate of 10 CFM until maximum antibioticyield was obtained.

B. Recovery The antibiotic of the present invention was recovered fromthe fennentation broth by adjusting the pH of the broth to 4.8 withhydrochloric acid and filtering. The filtered broth was passed through aDowex 50 X 2 sodium cycle resin (5 gallons) at a rate of 0.5 gallons perminute. The resin was washed with 10 gallons of water and eluted with 50gallons of 70 percent methanol, 30 percent lN ammonia at a rate of 0.25gallons per minute. Ten S-gallon cuts were taken and each wasneutralized to a pH 7 with 5 percent hydrochloric acid. The cuts wereassayed and the active cuts were evaporated to 7.5 gallons of water. Theconcentrate was adjusted to pH 8 and extracted three times with an equalvolume of chloroform, and the extracts were dried over sodium sulfate.The rich chloroform was passed through a column containing a mixture of70 percent Florisil, 30 percent Celite 545 at a 10 minute contact time.The absorption was followed with 10 gallons of chloroform wash followedby 50 percent chloroform-acetone elution at the same rate. 5% galloncuts of the eluate were taken and the cuts were assayed. The active cutswere combined and evaporated to 0.5 gallons of chloroform. Thechloroform concentrate was dried in a dish and taken up in about ml. ofmethanol. Ten volumes of ethyl ether were added and the insolublesfiltered. A 10 to 1 methanol-concentrated hydrochloric acid solution wasadded to the ether filtrate with stirring until no further precipitationoccurred. The precipitate was filtered, washed with ether and dried.

Methobottromycin of the present invention was isolated by partitionchromatography with 0.1 M pH 6.0 phosphate buffer as a developingsolvent. Celite impregnated with capryl alcohol was used as thestationary phase. The stationary phase is made by wetting 250 lbs. ofacid washed celite (diatomaceous earth) with a solution of 6 gallons ofcapryl alcohol and 24 gallons of acetone. The celite is air dried toremove acetone and packed in a satisfactory column in thin layers andtamping, to insure uniformity. After packing, the column is washed with0.1 M pH 6.0 phosphate buffer until one-half volume of buffer hasemerged. About 40 grams of the crude hydrochloride described above isdissolved in 8 gallons of pH 6.0 buffer and placed on the column. Thecolumn was developed with pH 6.0 buffer taking 5 gallon cuts. Eachfraction is assayed and examined by paper chromatography. The richfractions were worked up by concentration to about one-fifth volume,adjusted to pH 8.5 and extracted two times with an equal volume ofchloroform. The chloroform solutions were evaporated to dryness. Theresidual solids were dissolved in a small amount of methanol (about 500ml.) and diluted with 20 volumes of ethyl ether and filtered. To thefiltrate was added 1.2 N methanolic HCl until further addition caused noprecipitation.

The antibiotic HCl was collected and dried in vacuo with the followingresults:

Methobottromycin and its salts are valuable antibacterial agents which,as has been pointed out above, are active in inhibiting the growth ofvarious gram positive organisms. However, these antibiotics areextremely useful in the treatment of chronic respiratory disease ofchickens and infectious sinusitis of turkeys. In this embodiment of thepresent invention, it has been found that chronic respiratory disease ofchickens and infectious sinusitis of turkeys may be effectivelyinhibited by the use of methobottromycin or its salts which may beadministered by either the subcutaneous or the oral route. Furthermore,it has been found that methobottromycin and its salts are effective incontrolling chronic respiratory disease of chickens and infectioussinusitis of turkeys when administered in dosages ranging from about 0.1mgjkg. to about 250 mg./kg. of body weight of the bird without theresulting danger of toxicity and preferably from 0.5 mg./kg. to 150mg./kg. of body weight of the bird, depending on the route of therapy.

In order to illustrate the activity and the advantages of the presentinvention in utilizing methobottromycin as an antibiotic against chronicrespiratory disease of chickens and infectious sinusitis of turkeys thefollowing tests are given. It is understood, however, that they aregiven merely for the purpose of illustration and in no way are they tobe taken as limiting.

TEST 1 Activity of Methobottromycin Tartrate Salt Groups of 6-day oldwhite Leghorn chickens were employed in this test. Methobottromycintartrate was administered by the subcutaneous route. Doses of 2.0 to 100mg./kg. of bird were administered 18 and 25 hours after infection. Thebirds were infected by injecting intra-air sac a 72-hour old brothculture of Mycoplasma gallisepticum (PPLO), serotype A, strain 8-6 (fromH. E. Adler, University of CaIifornia-isolated from the brain of aninfected turkey. The test was terminated 4 weeks after infection and theresults were evaluated on the bases of mortality and body weight gain.

In the above test the body weight gain in the birds treated by themethod of the present invention showed significant increase over thoseof the infected controls. In addition, where 9 out of 12 or 75 percentof the infected controls died, only 1 out of 30 or 3 percent of thebirds treated by the method of the present invention died.

"nasrz Activity of Methobottromycin Free Base Groups of 6-day old whiteLeghorn chickens were employed in this test. Methobottromycin wasadministered by the subcutaneous route in doses of 0.8 to 4.0 mg./kg. ofbird, l8 and 25 hours after infection. The birds were infected via theair-sac route and the results evaluated by the same method as in Test 1above. The test was terminated after three weeks.

TABLE II Dose Mortal- Avg. wt. Sample mg/kg ity Gain-gm. Wt. Gain Nonnal0/6 207 Controls Infected 7/12 I I 8 57 Controls Metho 3 X 0.8 0/6 14872 bottromycin 3 X 2.0 2/6 158 76 3 X 4.0 0/6 177 86 In the above testthe body weight gain in the birds treated by the method of the presentinvention showed a significant increase over those of the infectedcontrols. In addition, where 7 administration) out of 12 or 58 percentof the infected controls died, only 2 out of 18 or 1 1 percent of thebirds treated by the method of the present invention died.

TEST 3 Activity of Methobottromycin Free Base Groups of six and I2chickens were employed in this test. Methobottromycin was administeredboth subcutaneously as shown in Table IIIA and orally as shown in TableIllB. In Test IIIA (subcutaneous adminstration) methobottromycin wasadministered 1, l8 and 25 hours after infection in dosages ranging from10 to mg./kg. of bird. In Test lllB (oral administration)methobottromycin was administered in ranges of from 5 to mg./kg. of bird1 hour after infection. This test was also terminated after 3 weeks. Thebirds were infected and the results evaluated by the same method as inTest 1 above.

In tests 111A and IIIB above the body weight gain in the birds treatedby the method of the present invention showed a significant increaseover those of the infected controls. In addition, in Test A(subcutaneous administration) where 7 out of 12 or 58 percent of theinfected controls died, none of the 48 birds treated by the method ofthe present invention died, showing 100 percent effective activity. Inthe Test B (oral administration) where 7 out of 12 or 58 percent of thecontrols died, only 3 out of 24 or 12 19 percent of the birds treated bythe method of the present invention died.

It should of course be understood that the theoretical explanations forthe possible formula and proportions of ingredients of methobottromycinas disclosed herein are based on our present knowledge of this productand does not exclude the possibility that subsequent experimental datawill establish that the postulated formula and proportions are, in fact,incorrect. Accordingly, it is not desired to be bound by thesetheoretical considerations, however likely they may appear to be in thelight of present knowledge. These explanations are presented principallyas a means for providing a better understanding of the presentinvention.

While specific embodiments of the present invention have been named anddescribed, it will be apparent to those skilled in the art that changesmay be made in the detail shown without departing from the spirit of thepresent invention or the scope intended. Any departure from the abovedescription which conforms to the present invention is intended to beincluded within the scope of the claims.

We claim:

1. A member of the group consisting of methobottromycin and its acidaddition salts, said methobottromycin being characterized by thefollowing properties:

a. Crystallizing in the form of white prisms melting at the temperatureof 166 to 167 C.;

b. Being soluble in alcohols, esters, ethers,

chlorinated solvents and benzene and partly soluble in water;

c. Having a specific rotation of [0111) 15 in a percent solution of 95percent ethanol;

d. Having an infrared absorption spectrum exhibiting characteristicpeaks at the following wave lengths expressed in reciprocal centimeters:3,300, 2,950, 1,730, 1,630-1,650, 1,490, 1,360, 1,300, 1,242, 1,160,1,132, 1,119, 1,102, 980, 806;

e. Containing the elements carbon, hydrogen,

nitrogen, sulfur, and oxygen in the following approximate proportions:

S 3.90% O 15.58% (by difference) Total 100.00% f. Having characteristicR, values in the following solvent systems:

n-butyl alcohol saturated with 1% aqueous acetic acid 0.85 n-butylalcohol saturated with 2% aqueous pyridine 0.84 ethyl acetate saturatedwith 1% aqueous acetic acid 0.37 ethyl acetate saturated with 0.1Mphosphate buffer (pH 7) 0.83 benzene:hexane:methanol:5% aqueous aceticacid (7:6: 10:6) 0.0 benzenezhexane:methanol:5% aqueous pyridine(7:6:l0:6) 0.5 benzene saturated with 1% aqueous acetic acid 0.0 benzenesaturated with 2% aqueous pyridine 0.5 capryl alcohol saturated with0.1M pH 6 phosphate buffer (reverse phase) 0.19

g. Yielding a hydrolysis with acid ninhydrin substances not includingproline.

2. Methobottromycin as defined in claim 1.

3. Acid addition salts of methobottromycin as defined in claim 1.

4. A process for the preparation of methobottromycin which comprisescultivating Streptomyces canadensis ATCC 17776 under aerobic conditionsin an aqueous nutrient medium until substantial antibiotic activity isimparted to said medium.

5. The process of claim 4 which comprises cultivating Streptomycescanadensis ATCC 17776 under aerobic conditions in a nutrient mediumcontaining soy bean meal, dextrose, distillers solubles, yeastautolysate, and tryptic digest of casein.

6. A method of treating poultry infected with pleuropneumonia-likeorganisms comprising administering to said infected poultry an effectivedose for inhibiting pleuropneumonia-like organisms of an antibioticselected from the group consisting of methobottromycin and its acidaddition salts as defined in claim 1.

7. The method of claim 6 wherein said effective dose is in the range offrom about 0.1 mg./kg. to about 250 mg./kg. of body weight of saidinfected poultry.

8. The method of claim 6 wherein said effective dosage of saidantibiotic is administered by subcutaneous route.

9. The method of claim 6 wherein said effective dosage of saidantibiotic is administered by oral route.

10. The method of claim 6 wherein the antibiotic administered ismethobottromycin.

11. The method of claim 6 wherein the antibiotic administered is an acidaddition salt of methobottromycin.

2. Methobottromycin as defined in claim
 1. 3. Acid addition salts ofmethobottromycin as defined in claim
 4. A process for the preparation ofmethobottromycin which comprises cultivating Streptomyces canadensisATCC 17776 under aerobic conditions in an aqueous nutrient medium untilsubstantial antibiotic activity is imparted to said medium.
 5. Theprocess of claim 4 which comprises cultivating Streptomyces canadensisATCC 17776 under aerobic conditions in a nutrient medium containing soybean meal, dextrose, distillers solubles, yeast autolysate, and trypticdigest of casein.
 6. A method of treating poultry infected withpleuropneumonia-like organisms comprising administering to said infectedpoultry an effective dose for inhibiting pleuropneumonia-like organismsof an antibiotic selected from the group consisting of methobottromycinand its acid addition salts as defined in claim
 7. The method of claim 6wherein said effective dose is in the range of from about 0.1 mg./kg. toabout 250 mg./kg. of body weight of said infected poultry.
 8. The methodof claim 6 wherein said effective dosage of said antibiotic isadministered by subcutaneous route.
 9. The method of claim 6 whereinsaid effective dosage of said antibiotic is administered by oral route.10. The method of claim 6 wherein the antibiotic administered ismethobottromycin.
 11. The method of claim 6 wherein the antibioticadministered is an acid addition salt of methobottromycin.